Figure 1
Expression of Brd in HUVECs and effect of BET bromodomain inhibition on angiogenesis.
(A) Brd mRNA expression in HUVECs. Brd mRNA expression was measured by qRT-PCR analysis. The data represent the mean (S.E.M.) from 3 independent experiments. (B) Effect of BET inhibition on VEGF-induced in vitro tube formation of HUVECs. HUVECs, which were transfected with or without specific Brd2 shRNA (shBrd2) or Brd4 shRNA (shBrd4) or control shRNA (shC), were suspended in serum-free medium and seeded onto the Matrigel. They were pretreated with or without dimethyl sulfoxide (DMSO, as a control) or various concentrations of JQ1 for 6 h and then stimulated with or without VEGF (10 ng/mL) for 6 h. The left panel shows representative images of decreased tube formation in JQ1 treatment compared with control cells from at least 3 independent experiments. The right panel shows quantitative analysis of the number of intact tubes from 3 microscopic fields per well (n = 4). (C) Effect of JQ1 on in vivo angiogenesis using a Matrigel plug assay. Matrigel (500 μL/plug) was injected subcutaneously into 7–8-week-old male null mice. Mice were then intraperitoneally treated daily with vehicle (DMSO) or JQ1 (50 mg/kg·d) for 10 d. The Matrigel plugs were embedded with paraffin. Next, 5 μm sections were stained with H&E, Masson’s trichrome stain (endothelial cells stain red and Matrigel stains blue), or CD34 immunohistochemistry. Photographs of representative Matrigel plugs show vessel growth and CD34 expression (left panel). n = 6 for each experimental group. The right panel shows quantitative analysis of CD34. (D) Effect of JQ1 (50 mg/kg·d, i.p) on retinal neovascularization in the retinopathy-of-prematurity model. Serial cross sections of eyes were stained with H&E and blood vessels in the retina were quantified by light microscopy under 400× magnification (right panel). Scale bars: 100 μm. All values represent mean ± SD. *P < 0.05 vs. DMSO or shC or Normoxia, #P < 0.05 vs. VEGF or VEGF + shC or DMSO.
